Abstract Japanese red pine Pinus densiflora is extensively cultivated in Japan, Korea, China, and Russia and is harvested for timber, pulpwood, garden, and paper markets. However, genetic information and molecular markers were very scarce for this species. In this study, over 51 million sequencing clean reads from P. densiflora mRNA were produced using Illumina paired-end sequencing technology. It yielded 83,913 unigenes with a mean length of 751 bp, of which 54,530 64.98% unigenes showed similarity to sequences in the NCBI database. Among which the best matches in the NCBI Nr database were Picea sitchensis 41.60%, Amborella trichopoda 9.83%, and Pinus taeda 4.15%. A total of 1953 putative microsatellites were identified in 1784 unigenes using MISA MicroSAtellite software, of which the tri-nucleotide repeats were most abundant 50.18% and 629 EST-SSR expressed sequence tag- simple sequence repeats primer pairs were successfully designed. Among 20 EST-SSR primer pairs randomly chosen, 17 markers yielded amplification products of the expected size in P. densiflora. Our results will provide a valuable resource for gene-function analysis, germplasm identification, molecular marker-assisted breeding and resistance-related genes mapping for pine for P. densiflora. View Full-Text